CZ20004216A3 - Specific binding molecules for scintigraphy, and conjugates containing such molecules - Google Patents

Abstract

The present invention relates to antibodies with sub-nanomolar affinity specific for a characteristic epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Furthermore, it relates to the use of radiolabeled high affinity anti ED-B antibodies for detecting new-forming blood vessels in vivo and a diagnostic kit comprising said antibody. Furthermore, it relates to conjugates comprising said antibodies and suitable photoactive molecules (e.g. a judiciously chosen photosensitizer), and their use for the selective light-mediated occlusion of new blood vesels.

Description

Technical field

The present invention relates to antibodies with subnanomolar affinity specific for the characteristic epitope of the ED-8 domain of fibronectin, which is a marker of angiogenesis. It also relates to the use of radiolabeled high-affinity anti-ED-8 antibodies for the detection of newly formed vessels in vivo and diagnostic kits containing said antibodies.

Furthermore, the invention relates to conjugates consisting of said antibodies and a suitable photoactive molecule (for example a photosensitizer) and their use in the detection and / or coagulation of newly formed blood vessels.

BACKGROUND OF THE INVENTION

Tumors cannot grow beyond a certain level without the formation of new blood vessels (without angiogenesis) and the correlation between microcapillary density and tumor invasiveness has been described for many tumors (Folkman, 1995, Nature Med. 1: 27-31). angiogenesis due to many ocular diseases that result in loss of vision (Lee et al., Surv. Opthalmol. 43: 245-269, 1988; Friedlander, M. et al., Proc. Natl. Acad. Sci. USA 93: 97649769, 1996). Molecules that can selectively target markers of angiogenesis will allow the diagnosis and therapy of tumors and other diseases characterized by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Angiogenesis markers are expressed in most aggressive solid tumors and should be readily accessible for intravenously administered specific ligands (Pasqualini et al., • 0).

1997, Nature Biotechnol. 15: 542-546; Neri et al., 1997,

Nature Biotechnol. 15: 1271-1275). Targeted occlusion of the neovasculature can lead to tumor infarction and collapse (O'Reilly et al., 1996), Nature Med. 2: 689-692; Huang et al., 1997, Science 275: 547-550).

The ED-B domain of fibronectin, a sequence of 91 amino acids identical in mice, rats and humans, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around the neovasculature (Castellani et al., 1994, Int. J. Cancer 59: 612- 618) and may represent a target for molecular intervention. Indeed, we have recently shown using fluorescence techniques that anti-ED-B single chain Fv antibody fragments (scFvs) selectively accumulate in tumor blood vessels in tumor-bearing mice and that the affinity of the antibody appears to determine the efficacy of targeted binding (Neri et al., 1997, Nature) Biotechnol 15: 1271-1275; International Patent Application No. PCT / GB97 / 01412, based on GB96 / 10967.3). Targeted tumor binding was evaluated 24 hours after injection or at later time intervals.

Various attempts are known in the art to obtain antibodies against the ED-B domain that could be used for targeted tumor binding.

Peters et al. (Cell Adhesion and Communication, 1995, 3: 67-89) describe polyclonal antibodies produced against antigens containing no sequence other than the intact EDB domain, and show that these antibodies bind directly and specifically to that domain.

However, the reagents described by Peters et al. they have a number of disadvantages: antiserum by Peters et al. recognizes ED-B (+) - FN • 4

Only after treatment with N-glycanase. 4 4 4 4 4 4 4 4 4 4 ® 444444 4 This renders these agents unsuitable for applications such as targeted tumor binding, imaging and therapy, since deglycosylation cannot be performed in vivo. The authors themselves report that their antibodies do not recognize complete ED-B (+) -FN produced by mammalian cells. They also report that it was impossible to produce monoclonal antibodies specific for the ED-B domain of fibronectin, although antibodies against other fibronectin domains (such as ED-A) were produced. It is well known in the art that polyclonal antibodies are unacceptable for the above applications.

Despite years of intensive research in this field, monoclonal antibodies recognizing the ED-B domain of fibronectin without N-glycanase treatment can only be produced using phage display techniques as used in the present invention.

Zang et al. (Matrix Biology, 1994, 14: 623-633) describe a polyclonal antiserum directed against a canine ED-B domain. The authors assume cross-reactivity with human ED-B (+) - FN, although this reactivity has not been tested. However, the authors describe difficulties associated with the production of monoclonal antibodies directly recognizing the fibronectin ED-B domain (page 631). The antiserum recognizes ED-B (+) - FN in Western blot only after N-glycanase treatment. As mentioned above, the need for glycanase treatment makes it impossible to use in the applications of the present invention.

Recognition of ED-B (+) - FN in ELISA proceeds without the need for deglycosylation, but only on the cartilage extracted with denaturing agent (4M urea) and captured on the plastic by gelatin. The authors report that the binding of the FN molecule to gelatin bound to

The plastic surface of an ELISA plate may in some way expose epitopes so that they can be recognized by antisera. Since FN cannot be denatured and bound to gelatin in in vivo applications, the monoclonal ligands of the present invention have distinct advantages.

Japanese patents JP02076598 and JP04169195 disclose anti-ED-B antibodies. It is not clear from these documents whether monoclonal anti-ED-B antibodies are described. In addition, it seems impossible for a single antibody (such as that described in JP02076598) to have an antigenic determinant in the amino acid sequence of formula (1), (2) or (3):

- (1) EGIPIFEDFVDSSVGY;

- (2) YTVTGLEPGIDYDIS;

- (3) NGGESAPTTLTQQT;

for the following reasons:

(i) the monoclonal antibody should recognize a well defined epitope;

(ii) the three dimensional structure of the ED-B domain of fibronectin was determined by NMR spectroscopy. The segments (1), (2) and (3) are located on opposite sides of the ED-B structure and cannot be bound simultaneously by one monoclonal antibody.

Furthermore, to demonstrate the usefulness of the antibodies, tumor localization as well as evidence of staining of EDB (+) -FN structures in biological samples without treatment with structure degrading agents should be reported.

The BC1 antibody described by Carnemolla et al., 1992, J. Biol. Chem. 267: 24689-24692, recognizes an epitope on FN domain 7, but not on the ED-B domain, which is cryptic in the presence of the ED-B domain of fibronectin. It is strictly human-specific. Therefore, the BC1 antibody and the antibodies of the present invention have different reactivity. D81e, the BC1 antibody recognizes domain 7 itself and fibronectin domain 7-8 in the absence of the ED-B domain (Carnemolla et al., 1992, J. Biol. Chem. 267: 24689-24692). Such epitopes may be produced in vivo by proteolytic degradation of FN molecules. An advantage of the agents of the present invention is that they can localize FN molecules or fragments only if they contain the ED-B domain.

For the diagnosis of tumors, and more precisely for imaging of primary and secondary tumor lesions, immunoscintigraphy is a method of choice. In this examination, patients are imaged by a suitable instrument (e.g., a gamma camera) after they have been injected with a radiolabeled compound (e.g., a radionuclide coupled to a suitable vehicle). For scintigraphic applications, short half-life isotopes that emit gamma radiation, such as technetium-99m, iodine-123, or indium-111, are typically used, minimizing patient exposure to ionizing radiation.

The most commonly used radionuclide in nuclear medicine departments is technetium-99m (99mTc), a gamma emitter with a half-life of 6 hours. Patients after injection of 99mTc-based radiopharmaceuticals can be imaged up to 12-24 hours after injection; however, accumulation of nuclide in the lesion earlier is desirable.

Further, if available antibodies were capable of rapid and selective localization of newly formed blood vessels, researchers would be stimulated to explore new suitable molecules for conjugating to antibodies to achieve a diagnostic and / or therapeutic effect.

• • • •

SUMMARY OF THE INVENTION

In view of the need for radiopharmaceuticals capable of localizing tumor lesions within hours of injection that exists in the nuclear medicine art, and considering that the affinity of the antibody appears to affect its efficacy in targeted detection of angiogenesis, the present invention provides the preparation of antibodies specific for the ED-B domain. fibronectin with a dissociation constant in the subnanomolar order (for a review of definitions and measurement of antigen-antibody affinity, see Neri et al., 1996, Trends in Biotechnol. 14: 465-470). Another object of the present invention are radiolabeled antibodies in a suitable format, directed against the ED-B domain of fibronectin, that detect tumor lesions as early as hours after injection.

In one aspect of the invention, these objects are achieved by an antibody with a specific affinity for a characteristic epitope of the ED-B domain of fibronectin and an improved affinity for said ED-B epitope.

In another aspect of the present invention, the antibody described above is used to rapidly target markers of angiogenesis.

Another aspect of the present invention is a diagnostic kit comprising said antibody and one or more agents for detecting angiogenesis.

Yet another aspect of the present invention is the use of said antibody for the diagnosis and therapy of tumors and diseases that are characterized by vascular proliferation.

Finally, an important aspect of the present invention are conjugates comprising said antibodies and suitable photoactive molecules (e.g., a properly selected photosensitizing agent), and their use for selective light-mediated occlusion of newly formed blood vessels.

Definition:

In the present invention, some terms have the following meanings.

The term antibody refers to an immunoglobulin, whether natural or wholly or partially synthetically prepared. The term also includes any polypeptide or protein having a binding domain that is an antibody binding domain or that is homologous to that domain. These may be obtained from natural sources or may be wholly or partially prepared synthetically. Examples of antibodies are immunoglobulin isotypes and subclasses of their isotypes; fragments that comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and dipribodies. It is possible to use monoclonal or other antibodies and use recombinant DNA technology techniques to produce other antibodies or chimeric molecules that retain the specificity of the parent antibody. Such techniques may include insertion of DNA encoding an immunoglobulin variable region or complementarity determining regions of immunoglobulins (CDRs), immunoglobulin constant regions, or constant regions plus framework regions of different immunoglobulins. See, for example, EP-A-184187, GB 2188638A or EP-A-239400. Hybridomas or other antibody-producing cells may be subjected to genetic mutations or other changes that may or may not alter the binding specificity of the antibodies produced. Because antibodies can

Φ φ φ φ φ «« «· · · · · · · φ · · · · · · φ φ · · · · · · · · φ φ φ φ φ Φ the term antibody includes any binding agents or substances that contain a binding domain with the desired specificity. Therefore, the term includes antibody fragments, derivatives, functional equivalents, and homologues, including any polypeptides comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetically prepared. Also encompassed by this term are chimeric molecules comprising an immunoglobulin binding domain, or equivalent thereof, fused to another polypeptide. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of whole antibody can act as binding antigens. Examples of binding fragments are (i) a Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of one antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 9: 341, 544-546) consisting of a VH domain; (v) isolated CDR regions; (vi) F (ab ') 2 fragments, bivalent fragments comprising two linked Fab fragments; (vii) single chain Fv molecules (scFV), wherein the VH domain and VL domain are linked by a polypeptide linker that allows the association of two domains to form an antigen binding site (Bird et al., (1988), Science 242: 423-426 Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83); (viii) bispecific single chain PV dimers (PCT / US92 / 09965) and (ix) dipribodies, multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; Holliger et al., (1993) Proc. Natl. Acad. Sci. USA) 90, 6444-6448).

Diproteins are multimers or polypeptides, wherein each polypeptide comprises a first domain consisting of an immunoglobulin light chain binding region and a second domain consisting of an immunoglobulin heavy chain binding region, wherein the two domains are linked (e.g., via a peptide linker).

0 0 0 • 0

0 0 0 0 0 0 0

(0 groups), but are not capable of associating with each other forming an antigen binding site; antigen binding sites are formed by associating a first domain of one polypeptide in a multimer with a second domain of another polypeptide in a multimer (WO 94/13804). When bispecific antibodies are used, conventional bispecific antibodies can be used, which can be prepared by various methods described in Holliger and Winter, 1993, Curr. Opin. Biotech., 4: 446-449, for example by chemical synthesis or from hybrid hybridomas, or any fragments of bispecific antibodies as mentioned above can be used.

It may be preferable to use scFv dimers or dipribodies than the use of whole antibodies. Dipribodies and scFvs can be prepared without the Fc region, using only variable domains, potentially reducing the effect of the anti-idiotypic response. Other forms of bispecific antibodies include single chain CRAbs as described in Neri et al., (1995) J. Mol. Biol. 246: 367-373.

The term complementarity determining regions (CDRs) of antibody variable domains refers to those hypervariable antibody sequences that contain residues necessary for specific antigen recognition. In this invention, we use the definition and numbering of CDRs given in Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917.

The term functionally equivalent variant refers to a molecule (variant) which, despite structural differences from another molecule (parent molecule), retains significant homology as well as at least some biological function of the parent molecule, for example the ability to bind to a particular antigen or epitope. Variants may be in the form of fragments, derivatives or mutants. Variants, derivatives, or mutants can be prepared by modifying the parent molecule by addition, deletion, substitution or insertion of one or more amino acids, or by binding to another molecule. These changes can be made at the nucleotide or protein level. For example, the co-polypeptide may be a Fab fragment that is then bound to the Fc terminal from another source. Alternatively, a marker such as an enzyme, fluorescein, etc. may be attached. For example, a functionally equivalent variant of antibody A against a characteristic epitope of the ED-B domain of fibronectin may be antibody B with a different complementarity determining sequence but recognizing the same epitope as antibody A.

We isolated recombinant antibodies in scFv form from an antibody phage display library that are specific for the ED-B domain of fibronectin and that recognize ED-B (+) - fibronectin in tissue sections. One of these antibodies, E1, was affinity-modified to obtain H10 and L19 antibodies with improved affinity. Antibody L19 has a dissociation constant for the ED-B domain of fibronectin at the order of subnanomolar concentrations.

The high affinity antibody L19 and D1.3 (antibody specific for an irrelevant antigen, chicken egg lysozyme) were radiolabeled and injected into tumor-bearing mice. Biodistribution in tumor, blood and organs was determined at different times and was reported as a percentage of injected dose per gram of tissue (% ID / g). As early as 3 hours after injection, L19% ID / g (tumor) was better than% ID / g (blood), but not for negative control D1.3. Tumor: blood ratios increased with later measurements. These data suggest that the high-affinity antibody L19 may be a useful tumor-targeting agent useful, for example, for immunoscintigraphic detection of angiogenesis.

• · »·

• 0 0 0 0 0 0 0 0 0 0 0 0

0 00

A photosensitizing agent can be defined as a molecule which, upon irradiation and in the presence of water and / or oxygen, produces toxic molecules (e.g. singlet oxygen) capable of reacting with biomolecules that potentially damage biological targets such as cells, tissues or body fluids. Photosensitizing agents are particularly advantageous if they absorb light at wavelengths above 600 nm. Indeed, light penetration in tissues and body fluids is maximal at 600-900 nm (Wan et al.,

1981, Photochem. Photobiol. 34: 679-681). Targeted administration of a photosensitizing agent followed by radiation is an attractive way to treat angiogenesis-related diseases (Yarmush, ML et al., Antibody Targeted Photolysis, Crit. Rev. Therap. Drug Carrier Systems, 10: 197252 (1993); Rowe, PM, Lancet 351 1496 (1998);

Trends Biotechnol. 13: 14-18 (1995)), in particular for selective ablation of ocular neovasculature. Available therapeutic modalities, such as laser photocoagulation, either directly or after administration of a photosensitizing agent, are limited by the lack of selectivity and usually result in damage to healthy tissues and vessels (Macular Photocoagulation Study Group, Arch. Opthalm. 112: 480-488, (1994; Haimovici, R. et al., Curr Eye Res. 16: 83-90 (1997), Schmidt-Erfurt, U. et al., Graefes Arch Clin Exp Opthalmol. 236: 365-374 (1998).

It is apparent from the arguments set forth above that it is extremely important to discover methods for improving the selectivity and specificity of photosensitizing agents, for example by conjugating them to suitable carriers. It is likely that the development of quality carriers will not be easy. In addition, it is likely that not all photosensitizing agents will be suitable for in vivo delivery to a selected site. Factors such as the chemical structure of a photosensitizing agent, its

Solubility, lipophilicity, adhesion and efficacy are likely to significantly affect the possibility of targeted administration and efficacy of conjugates containing photosensitizing agents.

We have shown that the high-affinity L19 antibody, specific for the ED-B domain of fibronectin, selectively locates newly formed blood vessels in a rabbit model of ocular angiogenesis after systemic administration. The L19 antibody, chemically coupled to the photosensitizing agent, a compound of tetravalent tin and chlorine e6 and irradiated with red light, causes selective occlusive neovasculature and induces apoptosis of the respective endothelial cells. These results demonstrate that new ocular vessels can be immunochemically differentiated from original ocular vessels in vivo and suggest that targeted administration of photosensitizing agents followed by radiation may be effective in treating ocular diseases causing blindness and perhaps other pathological conditions associated with angiogenesis.

Description of the figures in the attached drawings

Embodiments of the present invention are illustrated in the following drawings, wherein:

Giant. 1 shows the proposed antibody phage library;

Giant. 2 shows 2D gels and western blotting of human melanoma COLO-38 cell lysate;

Giant. 3A, 3B and 3C show immunohistochemical experiments on glioblastoma multiforme;

Giant. 4 shows stability analysis of antibody-(ED-B) complexes;

• 4 • 4 • 4 • 4 • 4

Giant. 5 shows the biodistribution of radiolabeled antibody fragments in tumor-bearing mice;

Giant. 6 shows the amino acid sequence of L19;

Giant. 7A and 7B show rabbit eyes with an implanted pellet;

Giant. 8 shows immunohistochemical staining of rabbit corneal sections;

Giant. 9 shows immunohistochemical staining of sections from rabbit eye structures (cornea, iris and conjunctiva) obtained using a red substrate for alkaline phosphatase and hernatoxylin;

Giant. 10 shows the location of fluorescently labeled antibodies in ocular neovasculature;

Giant. 11 shows the macroscopic appearance of the eyes of rabbits injected with a protein bound to a photosensitizing agent before and after irradiation.

Giant. 12 shows microscopic analysis of sections from ocular structures of rabbits injected with a protein bound to a photosensitizing agent and irradiated with red light.

Detailed description of the pictures

Giant. 1 shows the proposed antibody phage library.

(a) Antibody fragments are displayed on phage as pIII fusion as schematically indicated. In the antibody binding site (direct view of the antigen), the Vκ CDR scaffold is shown in yellow and the V H <·· • »

The CDR skeleton is shown in blue. The residues subjected to random mutagenesis are at positions 91, 93, 94 and 96 of the Vκ CDR3 (yellow) and at positions 95, 96, 97 and 98 of the VH CDR3 (blue). The Cb atoms of these side chains are represented by darker colors. Also shown are (gray) CDR1 and CDR2 residues that can be mutated to improve antibody affinity. Using RasMol (http://www.chemistry.ucsc.edu/wipke/teaching/rasmol.html) scFv structures were modeled from the pdb ligm array (Brookhaven Protein DataBank; http://www2.ebi.ac.uk/ pcserv / pdbdb.htm). (b) PCR amplification and cloning strategies for the library. DP47 and DPK22 germline templates were modified (see text) to generate mutations in the CDR3 regions. Genes are indicated by rectangles and CDRs are numbered boxes in genes. VH and VL segments were then assembled and cloned into pDN332 phagemid vector. The primers (from SEQ ID NO: 11 to SEQ ID NO: 18) used in the amplification are shown at the bottom of the figure.

Giant. 2 shows: (a) silver staining of 2D-PAGE lysate of human melanoma COLO-38 cells into which recombinant 7B89 containing ED-B was introduced. Two 7B89 spots (circled) are due to partial proteolysis of the His terminus used for protein purification; (b) immuno-transfer of a gel identical to that of Figure 2a using anti-ED-B E1 (Table 1) and M2 anti-FLAG antibodies as detection reagents. Only 7B89 spots are detected, confirming the specificity of the recombinant antibody isolated from the gel spot.

Giant. 3A-3C show immunohistochemical experiments performed on serial sections of glioblastoma multiforme, which show typical glomerulus-like vascular structures stained using scFV E1 (A), A2 (B) and G4 (C). Scale: 20 gm.

4 4 4 4

4 4 Μ 44 * 4

4 »

4 4 • 4 4 4

4 4

4 44

Giant. 4 shows the stability of antibody-(ED-B) complexes.

Analysis of scFV binding of E1, H10 and L19 to the ED-B domain of fibronectin. (a) BIAcore sensograms showing improved dissociation profiles obtained after affinity adjustments of the antibody; (b) Native gel electrophoresis of scFv- (ED-B) complexes. Only the high affinity antibody L19 can form stable complexes with a fluorescently labeled antigen. Fluorescence detection was performed as described (Neri et al., (1996), BioTechniques, 20:

708-712). (c) Competition of the scFv- (ED-B-biotin) complex with a 100-fold molar excess of non-biotinylated ED-B as monitored by electrochemoluminescence using an Origen instrument. A long half-life of the L19- (ED-B) complex was observed. Solid squares: L19; open triangles: H10.

Giant. 5 shows the biodistribution of radiolabeled antibody fragments in tumor-bearing mice. Tumor and blood biodistribution, expressed as a percentage of injected dose per gram, are plotted against time. Relevant organ biodistribution is also listed.

Giant. 6 shows the amino acid sequence of antibody L19 comprising a heavy chain (VH), a linker, and a light chain (VL).

Giant'. 7A and 7B show rabbit eyes with implanted polymer pellets dipped in an angiogenic substance.

Giant. 8 shows immunohistochemical staining of rabbit corneal sections with newly formed vessels, using the L19 antibody.

Giant. 9 shows immunohistochemical staining of sections from eye structures using the L19 antibody. Specific red coloration is evident by the round of neovascular structures in the cornea (a), but not • · ···

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0

Ο 0

0 0

0 0

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0 0 * • 0 0 «0 1

0 0 0 0 0

Around the blood vessels in the iris (b) and the conjunctiva (c). Small arrows: corneal epithelium. Relevant blood vessels are indicated by large arrows. Scale bar: 50 μιη.

Giant. 10 shows immunophotodetection of fluorescently labeled antibodies detecting ocular angiogenesis. Strong fluorescent corneal neovascularization (indicated by arrows) is observed in rabbits injected with the L19Cy5 (a) antibody conjugate specific for the ED-B domain of FN, but in rabbits injected with the antibody conjugate HyHEL-10-Cy5 (b) . Immunofluorescence microscopy of corneal sections confirmed that L19-Cy5 (c) but not HyHEL-Cy5 (d) occurs around the corneal neovasculature. Sections (a, b) were obtained 8 hours after antibody injection; (c, d) were obtained using corneal sections isolated from rabbits 24 hours after antibody injection. P, pellet.

Giant. 11 shows the macroscopic appearance of the eyes of rabbits receiving conjugates containing a photosensitizing agent. The eye of a rabbit given L19-PS before (a) and 16 hours after (b) irradiation with red light. The arrow indicates coagulated neovasculature, which was confirmed as a hypofluorescent region in the Cy5 fluoroangiogram in Figure (c) 16 hours after irradiation. Note that no coagulation is observed in other vascular structures, such as dilated conjunctival vessels. For comparison, a fluoroangiogram with hyperfluorescence of leaking blood vessels is shown and a corresponding color photograph of the untreated rabbit eye is shown in (d) and (h). Giant. (e, f, g) are analogous to (a, b, c), but are for rabbits injected with ovalbumin-PS and who have been irradiated with red light. No coagulation was observed and the angiogram showed hyperfluorescence of leaking blood vessels. Eyes of rabbits in early stage of angiogenesis given L19-PS, 9 9 9 9 9 9 9

9 9 9 9 9

999 99 »·· 999 99 9t are shown on (i — 1). Imaging before (I) and 16 hours after irradiation with red light (j) showed significant and selective light-induced intravascular coagulation (arrow). The occlusion of the blood vessels (arrow) is particularly pronounced in the irradiated eye (1) of the rabbit immediately after euthanasia, but is not detectable in the non-irradiated eye (k) of the same rabbit. P, pellet. The tips indicate the corneo-scleral transition (limbus). In all figures, pre-existing dilated conjunctival vessels are visible above the limbus, while growth of the corneal neovasculature can be observed from the limbus to the pellet (P).

Giant. 12 shows a microscopic analysis of a selective occlusion of blood vessels. H / E corneal sections (a, e, b, f: not fixed; i, j: fixed with paraformaldehyde) of rabbits injected with ovalbumin-PS (a, e, i) or L19-PS (b, f, j) and who were exposed. Large arrows represent intact (e, i) or fully enclosed (f, j) blood vessels. In contrast to selective occlusion of corneal neovasculature and limited perivascular involvement (eosinophilia) caused by L19-PS after irradiation (b, j, f), blood vessels in the conjunctiva (k) and iris (1) show no signs of injury in the same rabbit. The fluorescent TUNEL assay showed different numbers of apoptotic cells in sections from irradiated rabbits receiving L19-PS (c, g), or ovalbumin-PS (d, h). Large arrows indicate some relevant vascular structures. Small arrows show corneal epithelium. Scale: 100 μιη (a-d) and 25 μπι (e-1).

The invention will now be described in more detail in the following examples.

• ·

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Isolation of human scFv antibody fragments specific for the ED-B domain of fibronectin from an antibody phage display library

The human antibody library was cloned using VH (DP47; Tomlison et al., (1992), J. Mol. Biol., 227: 776-798) and Vk (DPK22; Cox et al., (1994), Eur. J). Immunol. 24: 827-836) germline genes (for cloning and amplification strategies, see

1). The VH component of the library was created using partially. degenerate primers (FIG. 1) (from SEQ ID NO: 11 to SEQ ID NO: 11)

NO: 18) using a PCR-based technique for insertion of random mutations at positions 95-98 (CDR3). The VL component of the library was prepared in the same manner by inserting random mutations at positions 91, 93, 94 and 96 of CDR3. PCR reactions were performed as described (Marks et al., (1991), J. Mol. Biol. 222: 581-597). VH-V1 scFv fragments were prepared by PCR assembly (Fig. 1; Clackson et al., ( 1991), Nature 352: 624-628) from gel-purified VH and VL segments. 30 gg of purified VH-VL scFv fragments were digested twice with 300 units of NcoI and NotI, then the obtained material was ligated into 15 gg of NotI / NcoI digested pDN332 phagemid vector. pDN332 is a phagemid derivative of pHEN1 (Hoogenboom et al., (1991), Nucl. Acids Res. 19: 4133-4137) in which the sequence was between the NotI site and the amber codon before

gene III replaced by the following sequence encoding D3SD3-FLAG His6 ending 385-390). (Neri et al., (1996), Nature Biotechnology 14: Notl D D D S D D D Y K D D

5 '- GCG GCC GCA GAT GAC

DDKHHHHHH amber GAC GAC CAT CAT CAT CAT CAT CAT TAG - 3 '• · ·· ··

Transformations into the TG1 strain of E. coli were performed according to the method of Marks et al. (1991, J. Mol. Biol. 222: 581-597) and phages were prepared according to standard protocols (Nissim et al., (1991), J. Mol. Biol. 222: 581-597). Five clones were randomly selected and sequenced to verify the absence of contamination.

Recombinant fibronectin fragments of ED-B and 7B89, containing one and four type III homologous repeat sequences, respectively, were expressed from pQE12-based expression vectors (Qiagen, Chatsworth, CA, USA) as described in the literature (Carnemolla et al., 1992, Int. J. Cancer 68: 397-405).

Selections against the recombinant ED-B domain of fibronectin (Carnemolla et al., 1992, Int. J. Cancer 68: 397-405; Zardi et al., 1987, EMBO J., 6: 2337-2342) were performed at 10 nM concentration. using a biotinylated biotin disulfide-N-hydroxysuccinimide ester antigen (reagent B-4531; Sigma, Buchs, Switzerland; 10) and eluted from a 2D gel, and streptavidin-coated Dynabeads beads (Dynal, Oslo, Norway). For each round of paneling, 1013 phages were used per ml of reaction mixture. The phages were incubated with antigen in 2% milk / PBS (MPBS (for 10 minutes. To this solution was added 100 μΐ Dynabeads (10mg / ml; Dynal, Oslo, Norway) pre-blocked in MPBS. After 5 minutes of mixing, the beads Magnetically separated from the solution and washed 7 times with PBS-0.1% Tween20 (PBST) and 3 times with PBS, eluting by incubation for 2 minutes with 500 μΐ 50 mM dithiothreitol (DTT) to reduce disulfide bridges between antigen The beads were recovered and the solution was used to infect exponentially growing E. coli TG1 cells, and after three rounds of paneling, the eluted phage was used to infect exponentially growing E. coli HB2151 cells, which were placed on (2xTY • · · · · + 1% glucose + 100 gg / ml ampicillin) - 1.5% agar plate Individual colonies were cultured in 2xTY + 1% glucose + 100 gg / ml ampicillin and were induced overnight at 30 ° C with 1 mM IPTG to achieve production The supernatant formed Ants were screened by ELISA using streptavidin-coated microtiter plates treated with 10 nM biotinylated ED-P and anti-FLAG M2 antibody (IBI Kodak, New Haven, CT) as detection reagent. 32% of the colonies examined were positive in this assay and three of them that had the strongest ELSA signal (E1, A2 and G4) were sequenced and further characterized.

ELISA assays were performed using gel-stained biotinylated ED-B, non-denatured biotinylated ED-B, ED-B bound to adjacent fibronectin domains (recombinant protein containing 7B89 domains), and many irrelevant antigens. The E1, A2 and G4 antibodies reacted strongly and specifically with all three ED-B containing proteins. This, together with the fact that all three recombinant antibodies could be purified from bacterial supernatants using an ED-B affinity column, strongly suggests that they recognize an epitope present in the natural conformation of ED-B.

No response was detected for fibronectin fragments lacking the ED-B domain (data not shown). To test whether the gel-stained antibodies have good affinity for the natural antigen, real-time interaction analysis was performed using surface plasmon resonance on a BIAcore instrument, according to a method reported in the literature (Neri et al., (1997), Biotechnol Nature). 15: 12711275). The monomeric fractions of the E1, A2 and G4 scFv fragments bound to ED-B with an affinity in the range of 107-108 M-1 (Table 1).

As another assay for antibody specificity and usability, a 2D-PAGE immunoassay was performed, in which a human melanoma cell line COLO-38 lysate was introduced into the gel to which minimal amounts of 7B89 protein containing ED-B were added (Fig. 2). ScFv (E1) stained strongly and specifically only the 7B89 stain.

Antibodies E1, A2 and G4 were used for immunolocalization of fibronectin containing ED-B (B-FN) in cryostat sections from glioblastoma multiforme, an aggressive brain tumor with marked angiogenesis. Giant. 3 shows serial sections of glioblastoma multiforme, with typical glomerulus-like vascular structures stained red by three antibodies. Immunostaining of sections from glioblastoma multiforme samples frozen immediately after surgery was performed as described in the literature (Carnemolla et al., 1992, Int. J. Cancer 68: 397-405; Castellani et al., 1994, Int. J. Cancer 59: 612618). Briefly, immunostaining was performed using M2-antiFLAG antibody (IBI Kodak), biotinylated anti-mouse polyclonal antibodies (Sigma), a staining kit containing a streptavidin-biotin-alkaline phosphatase complex (BioSpa,

Milan, Italy) and naphthol-AS-MX-phosphate and fast red TR (Sigma). Gill hematoxylin was used as a contrast stain, followed by placement in glycergel (Dako, Carpenteria, CA) as previously described (Castellani et al., 1994, Int. J. Cancer 59: 612-618).

Using a similar technique and L19 antibody (see another example), we could also specifically stain newly formed vessels induced by implantation of polymer pellets immersed in an angiogenic substance, such as vascular endothelial growth factor or phorbol esters, into rabbit cornea.

Example 2: Isolation of human scFv antibody fragment binding to ED-B with subnanomolar affinity

• ·

ScFv (E1) was selected to test the possibility of improving its affinity by a limited number of mutations of CDR residues located on the periphery of the antigen binding site (Fig. IA). Combinatorially, we mutated residues 31-33, 50, 52 and 54 of the VH antibody and displayed the appropriate repertoire on filamentous phage. These residues often occur in contact with the antigen in the known 3D-structures of the antibody-antigen complexes. The obtained repertoire of 4 χ 10 ⁸ clones was selected for binding to the ED-B domain of fibronectin. After two rounds of paneling and after screening 96 individual clones, an antibody with 27-fold improved affinity (H10; Tables 1 and 2) was isolated. Consistent with other observations for affinity-engineered antibodies, we observed that improved affinity was due to slower dissociation from the antigen rather than improved con-values (Schier et al., (1996), Gene, 169: 147-155; Ito (1995, J Mol. Biol. 248: 729-732) It is believed that the light chain of the antibody contributes less to the binding affinity for the antigen, supported by the fact that both natural and synthetic antibodies without light chain can still bind to the antigen ( Ward et al., 1989, Nature 341: 544-546; Hamers-Casterman et al., 1993, Nature 363: 446-448) For this reason, we selected only two residues (32 and 50) of the VL domain for randomization that are located centrally in the antigen-binding site (Fig. 1a) and are often in contact with the antigen in 3D structures.The obtained library containing 400 clones was displayed on phage and was selected for antigen-binding. interaction profiles using real-time analysis performed using the BIAcore instrument (Jonsson et al., 1991, BioTechniques 11: 620-627) and measuring the _O ff by competition experiments with electrochemiluminescent detection was identified clone (L19), which is bound the ED-B domain of fibronectin with K d = 54 pM (Tables 1 and 2). Affinity treatment experiments were performed as follows: The scFv (E1) gene was amplified.

PCR using primers LMBlbis (5'-GCG GCC CAG CCG GCC ATG GCC GAG-3 ') (SEQ ID NO: 1) and DP47CDR1for (5'-GA GCC TGG CGG ACC CAG ATM NNM NNM NNGCTA AAG GTG AAT CCA GAG GCT G-3 ') (SEQ ID NO: 2) to insert random mutations at positions 31-33 of CDR1 VH (for numbering: 28), and with primers DP47CDRlback (5'-ATG AGC)

TGG GTC CGC CAG GCT CC-3 ') (SEQ ID NO: 3) and DP47CDR2for (5'GTC TGC GTA GTA TGT GGT ACC MNN ACT ACC MNN AAT MNN TGA GAC

CCA CTC CAG CCC CTT-3 ') (SEQ ID NO: 4) for random mutation of positions 50, 52, 54 in CDR2 VH. The remaining scFv gene fragment, within the 3'-part of the VH gene, peptide linker and VL gene, was amplified using primers DP47CDR2back (5'-ACA)

TRAY TAC GCA GAC TCC GTG AAG-3 ') (SEQ ID NO: 5) and JforNot (5 TCA TTC TCG ACT TGC GGC CGC TTT GAT TTC CAC CTT GGT CCC TTG GCC GAA CG-3 ') (SEQ ID NO: 6) (94 ° C) 1 minute, 60 ° C, 1 minute, 72 ° C, 1 . minute). Three obtained PCR products were

gel purified and PCR (21) was prepared using primers LMBlbis and JforNot (94 ° C, 1 minute, 60 ° C, 1 minute, 72 ° C, 1 minute). The obtained single PCR product was purified from the PCR mixture, was digested twice with NotI / NcoI and ligated into the NotI / NcoI digested vector pDN332. Approximately 9 µg of vector and 3 µg insert were used in the ligation mixture, the mixture was purified by phenolization and ethanol precipitation, resuspended in 50 µΐ sterile water and electroporated into electrocompetent E. coli TGI cells. The obtained affinity-engineered library contained 4 x 10 clones. Antibody-phage particles obtained as described (Nissim et al., 1994, EMBO J., 13: 692-698) were used for the first round of selection on a 7B89-coated immunotube (Carnemolla et al., 1996, Int. J. Cancer 68). : 397-405). The selected phages were used for a second round of paneling performed with biotinylated ED-B followed by capture on streptavidin-coated magnetic beads (Dynal, Oslo, Norway; see previous paragraph). After selection, approximately 25% of the clones were positive in the soluble ELISA (for the protocol of the experiment see previous chapter). From the candidates positive in ELISA, we further identified the one clone (H10; Table 1) with the lowest value of _O ff by BIAcore analysis (Jonsson et al., 1991, BioTechniques 11: 620-627).

The scFV (H10) gene was amplified by PCR using primers LMBlbis and DPKCDR1for (5'-G TTT CTG CTG GTA CCA GGC TAA MNN GCT GCT GCT AAC ACT CTG ACT G-3 ') (SEQ ID NO: 7) to insert random mutations to position 32 in CDR1 VL (for numbering:

Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917), and with the primers DPKCDRlback (5'-TTA GCC TGG TAC CAG CAG AAA CC-3 ') (SEQ ID NO: 8) and DPKCDR2for (5'-GCC AGT GGC CCT GCT GGA TGC MNN ATA GAT GAG GAG CCT (GGG AGC C-3 ') (SEQ ID NO: 9) to insert a random mutation at position 50 in CDR2 VL. The remaining portion of the scFv gene was amplified using DPKCDR2back oligonucleotides (5'-GCA TCC AGC AGG GCC ACT GGC-3 ') (SEQ ID NO:

10) and JforNot (94 ° C, 1 minute, 60 ° C, 1 minute, 72 ° C, 1 minute). The three obtained PCR products were assembled, digested, and cloned into pDN332 as described above for heavy chain mutagenesis. The obtained library was incubated with biotinylated ED-B in 3% BSA for 30 minutes and then captured on a streptavidin-coated microtiter plate (Boehringer Mannheim GmbH, Germany) for 10 minutes. Phages were eluted with 20 mM DTT solution (1,4-dithio-DL-threitol, Fluka) and were used to infect exponentially growing TG1 cells. Analysis of the binding of supernatant from 96 colonies to ED-B by ELISA and BIAcore allowed identification of clone L19. Anti-ED-B E1, G4, A2, H10 and L19 scFv antibody fragments selectively stained newly formed vessels in sections from aggressive tumors (Fig. 3).

The above anti-ED-B antibody fragments were then produced by inoculating one fresh colony into 1 liter of 2xTY.

The media as described in the literature (Pini et al., 1997, J. Immunol. Meth. 206: 171-182) and were affinity purified on a CNBr activated sepharose column (Pharmacia, Uppsala,

Sweden) in which 10 mg of 7B89 recombinant protein containing ED-B was bound (Carnemolla et al., 1996, Int. J. Cancer 68: 397-405). After loading, the column was washed with 50 ml of equilibration buffer (PBS, 1 ml EDTA, 0.5 M NaCl). The antibody fragments were then eluted with 100 mM triethylamine, immediately neutralized with 1 M Hepes, pH 7, and dialyzed against PBS. Affinity measurements with BIAcore were performed with purified antibodies as described in the literature (Neri et al., 1997, Nature. Biotechnol. 15: 1271-1275) (Figs.

4). Stripe shift analysis was performed as described in the literature (Neri et al., 1996, Nature. Biotechnol. 14: 385390), using recombinant ED-B fluorescently labeled at the N-terminus (Carnemolla et al., 1996, Int. J. Cancer 68: 397-405; Neri et al., 1997, Nature, Biotechnol. 15: 1271-1275) by the infrared fluorophore Cy5 (Amersham) (Fig. 4). BIAcore analysis does not allow accurate determination of kinetic parameters for slow dissociation reactions, due to possible reconnection, baseline instability and long measurement times necessary to determine that the dissociation phase proceeds according to one exponential profile. We therefore performed measurements of the kinetic dissociation constant of _O ff by competition experiments (Neri et al., 1996, Trends in Biotechnol. 14: 465-470) (Fig. 4). Briefly, anti-ED-B antibodies (30 nM) were incubated with biotinylated ED-B (10 nM) for 10 minutes, in the presence of M2 anti-FLAG antibody (0.5 μρ / ιηΐ) and polyclonal anti-mouse IgG (Sigma) which was previously labeled with a rutenium complex as described in the literature (Deaver, DR, 1995, Nature 377: 758-760). Non-biotinylated ED-B (1 μΜ) was added to this solution at different times in parallel reactions. Then streptavidin-coated Dynabeads, diluted in

• 0 • 0 ·

000

0 0 · 0 «00

Origen Assay Buffer (Deaver, D.R., 1995, Nature 377: 758-760) (20 μΐ, 1 mg / ml) and the mixtures obtained were analyzed using an ORIGEN analyzer (IGEN Inc, Gaithersburg, MD, USA). This instrument detects an electrochemoluminescence signal (ECL) that correlates with the amount of scFV fragment that remains bound to biotinylated ED-B at the end of the competition reaction.

The ECL signal versus competition time graph gives a profile that can be adjusted for a single exponential curve with a characteristic constant k ₀ ff (Fig. 4, Table 2).

Example 3: Tumor targeting with high affinity radiolabeled scFv specific for the ED-B domain of fibronectin

Iodine-labeled scFv (L19) or scFv (D1.3) (an irrelevant antibody specific for hen egg lysozyme) was injected intravenously into mice with subcutaneously implanted mouse F9 teratocarcinoma, a rapidly growing aggressive tumor. Antibody biodistribution was determined at different times (Fig. 4). ScFv (L19) and scFv (D1.3) were affinity purified on an antigen column (Neri et al., 1997, Nature Biotechnol. 15: 1271-1273) and radiolabeled with iodine 125 using the Iodogen method (Pierce, Rockford, IL, USA). The radiolabeled antibody fragments retained> 80% immunoreactivity as determined by loading the radiolabeled antibody into the antigen column, followed by radioactivity determination in the first and second effluent fractions. Nude mice (Swiss nude 12 weeks old, male) with subcutaneously implanted F9 mouse teratocarcinoma column (Neri et al., 1997, Nature Biotechnol. 15: 1271-1273) were injected with 3 gg (3-4 μθί) scFv at 100 μΐ saline solution. Tumor sizes were 50-250 mg because larger tumors tend to develop a necrotic center. However, when trying to focus

Φ •

4 4444 4 · 9 Φ Φ 9 4 • 4 Φ «· * ♦ Φ Φ · ΦΦ 49 • 444 99

• «« •

Nád larger tumors (300-600 mg) achieved essentially the same results. Three animals were used for each time. Mice were sacrificed humanely and their organs weighed and their radioactivity determined. Results for representative organs are expressed as a percentage of injected antibody dose per gram of tissue (% ID / g). ScFv (L19) is rapidly eliminated from the blood by the kidneys; it does not accumulate in the liver or other organs compared to conventional antibodies. 8% of the injected dose per gram of tissue is localized in the tumor as early as three hours after injection; the subsequent decrease in this value is due to the fact that the tumor doubles in size within 24-48 hours. The tumor: blood ratios at 3, 5, and 24 hours after injection are 1.9, 3.9, and 11.8, respectively, for L19, but always less than 1.0 for the negative control antibody.

Radiolabelled scFv (L19) is preferably localized in tumors as early as several hours after injection, indicating its utility for immunoscintigraphic detection of angiogenesis in patients.

Example 4: Anti-ED-B antibodies selectively stain newly formed ocular blood vessels.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a characteristic process that causes many diseases, including tumors and most eye diseases leading to vision loss. The ability to selectively recognize and occlude neovasculature will bring new diagnostic and therapeutic options.

We examined whether B-FN is a specific marker of ocular angiogenesis and whether antibodies recognizing B-FN can selectively recognize ocular neovascular structures in vivo after • 0

0

0 systemic administration. For this purpose, we stimulated angiogenesis in rabbit cornea that allowed direct observation of newly formed ocular vessels by surgical implantation of pellets containing vascular endothelial growth factor or phorbol ester (Fig. 7). Sucralfats (kind gift from Merck,

Darmstad, Germany) / Hydron pellets containing either 800 ng of vascular endothelial growth factor (Sigma) or 400 ng of phorbol-12-myristate, 13-acetate (PMA, Sigma) were implanted in the cornea of female New Zealand White rabbits, as described in the literature. (D'Amato, RJ et al., Proc. Natl. Acad. Sci. USA 91: 4082-4085, 1994). Angiogenesis was induced by both factors. Rabbits were watched daily. Using both inducing agents, newly formed blood vessels were strongly positive for ED-B in immunohistochemistry. PMA pellets were used for all further experiments. Immunohistochemical investigations revealed that L19 strongly stained neovasculature induced in rabbit cornea (Figs. 8, 9a), but not pre-existing blood vessels of the eye (Figs. 9b, c) and other tissues (data not shown). Immunohistochemistry was performed as described in the literature (Carnemolla, B. et al., Int. J. Cancer, 68: 397-405, 1996).

Example 5: Human antibody fragment L19, which binds to ED-B with subnanomolar affinity, recognizes ocular angiogenesis in vivo

Using the rabbit corneal angiogenesis model described in the previous example and immunophotodetection techniques (Neri, D. et al., Nature Biotechnol. 15: 1271-1275, 1997), we have shown that L19, chemically bound to a red fluorophore Cy5, but not the antibody fragment (HyHEL-10) -Cy5, directed against irrelevant antigens (Fig. 10a, b), selectively recognizes ocular angiogenesis after intravenous injection.

·· ·· »· · <

Fluorescent staining of growing ocular vessels was clearly detectable with L19 immediately after injection and persisted for at least two days, analogous to previous observations for tumor angiogenesis. Subsequent ex vivo immunofluorescence microscopic analysis of corneal sections confirmed the localization of L19, but not HyHIEL-10, around vascular structures (Fig. 10c, d). The demonstration of recognition of ocular neovascularization by an antibody, together with the reactivity of anti-B-FN antibodies in different species, encourages further clinical research. Immunofluorescence imaging may be useful for early detection of ocular angiogenesis in patients at risk before lesions become manifest in fluoroangiography.

Some methodological details:

For ex vivo immunofluorescence and for some H / E staining, the corneas were fixed in 4% paraformaldehyde in PBS before immersion. Fluorescence photodetection experiments were performed on rabbits calmed with Acepromazine at 5 mg / kg. For the targeted recognition experiments, 3.5 mg scFv (L19) i-Cy50, 66 and 2.8 mg scFv (HyHEL-10) i-Cy50, 83 were injected intravenously into each rabbit (injection time = 15 minutes).

Strong fluorescence in corneal neovasculature was observed immediately after injection of L19 but not HyHEL-10, and persisted for several hours. In another specificity assay, rabbits were injected the day before with scFv (HyHEL-10) -Cy5, and photodetection negative rabbits were injected the next day with scFv (L19) -Cy5, and strong fluorescent staining of the corneal newly formed vessels was observed.

For fluorescence detection, the eyes were irradiated with a tungsten halogen lamp (model Schott KL1500; Zeiss, Jena, Germany) equipped with a Cy5 excitation filter (Chromá, Brattleboro, VT).

USA) and two light channels whose ends were located approximately 2 cm from the eye. Fluorescence was detected with a cooled C-5985 monochrome CCD camera (Hamatsu, Hamatsu-City, Japan), equipped with a C-mounted Canon Zoom lens (V6x16; 16-100 mm, 1: 1.9) and a Cy5 emission filter with a 50 mm diameter (Chromá), placed 3-4 cm from the irradiated eye. Acquisition times were 0.4 sec.

Cy5 fluoroangiographic experiments were performed with the same equipment but with an intravenous injection of 0.25 mg Cy5-Tris (reaction product between Cy5-NHS and tris (hydroxymethyl) aminomethane; injection time = 5 minutes). Acquisition times were 0.2 s.

The antibody fragments were in the form of scFv. Purification of scFv (L19) and scFv (HyHEL-10) and their labeling with N-hydroxysuccinimide esters (NHS) of indolkyanine has been described in the literature (Neri, D. et al., Nature Biotechnol. 15: 1271-1275, 1997; Fattorusso, R. et. al., 1999, Structure 7: 381-390). The antibody: Cy5 ratios for labeling for the two antibodies were 1.5: 1 and 1.2: 1, respectively. Cy5-NHS was purchased from Amersham Pharmacia Biotech (Zurich, Switzerland) and ovalbumin from Sugma (Buchs, Switzerland).

After labeling, antibody conjugates were separated from unincorporated fluorophore or photosensitizer using PD-10 columns (Amersham Pharmacia Biotech) equilibrated in 50 mM phosphate, pH 7.4, 100 mM NaCl, (PBS). Immunoreactivity of antibody conjugates was measured by affinity chromatography on antigen columns (Neri, D. et al.,

Nature Biotechnol. 15: 1271-1275, 1997) and was> 78% in all cases. Immunoconjugates were analyzed by electrophoresis for:

A.:. .:. Sodium dodecyl sulphate-polyacrylamide gels and migrated as a band of MW = 30000 Daltons (purity ³ 90%).

Example 6: Human antibody fragment L19, chemically conjugated to a photosensitizing agent, which was tetravalent tin chlorine e _6r, selectively recognizes angiogenesis and occludes newly formed vessels under red light irradiation

To test whether selective vascular ablation can be achieved with selective antibody recognition, we injected L19 rabbits with an antibody fragment or irrelevant protein that does not trap in newly formed vessels (ovalbumin) bound to a photosensitizing agent that was tetravalent tin chloride ββ, ( referred to herein as PS). The eyes of injected animals were irradiated with red light (dose = 78 J / cm ² ). Representative values are shown in Fig. 11. A striking macroscopically different finding was observed 16 hours after irradiation in L19-PS-treated rabbits (Figs. 11a, b), with coagulation of the corneal neovasculature but not the conjunctival or other ocular structures. Cyoro-fluorine fluorophiography (Fig. 11c) confirmed vascular occlusion, which manifested itself as characteristic hypofluorescent regions. Conversely, hyperfluorescent regions were observed in transmissive neovasculature in unirradiated eyes (Fig. 11d, h). No macroscopic alterations were observed in the irradiated vessels of ovalbumin PS-treated rabbits (Fig. 11le-g), either ophthalmoscopically or in Cy5 fluororoangiography. The effect of irradiation of the targeted L19-PS conjugate in the early stages of corneal angiogenesis is shown in Fig. 11-1. Selectively coagulated blood vessels were macroscopically visible in living ··

animals (Fig. 11, j) and were even more pronounced in animals immediately after sacrifice (Fig. 11k, 1).

Photodynamic damage was further examined using microscopic techniques. After irradiation, vascular occlusion could be detected by standard hematoxylin / eosin (H / E) staining techniques in both unfixed and paraformaldehyde-fixed corneal sections from L19-PS-treated animals (Fig. 11b, j, f) but not ovalbumin-treated animals. PS (Fig. 11a, e, i). Apoptosis in the portion of the cornea targeted by the conjugate containing the photosensitizing agent was clearly visible in the fluorescent TUNEL assay (Fig. 11c, g), but was poorly detectable in the negative controls (Fig. 11d, h). Higher magnification showed apoptosis of endothelial cells in vascular structures (Fig. Lig). No damage to the blood vessels in the iris, sclera or conjunctiva of the treated animals was detected in either the TUNEL assay (not shown) or the H / E staining (Fig. 11k, 1).

Selective photodynamic ablation of neovasculature will be beneficial for the treatment of ocular diseases and other angiogenesis-related pathological conditions that are accessible for irradiation with light diffusers or optical fibers. The results of this study clearly show that ocular neovasculature can be selectively occluded without damaging pre-existing blood vessels and normal tissues.

Some technical details:

The tetravalent tin and chlorine compound e6 was selected from various photosensitizing agents for its potency, solubility and specificity after binding to rabbit anti-mouse polyclonal antibody (Sigma). These immunoconjugates were tested for targeted photolysis of red blood cells coated with a monoclonal antibody specific for human CD47 (SEQ.

· »» · «·»

313441A; Pharmingen, San Diego, CA). The tetravalent tin and chlorine compound e6 was prepared as described in the literature (Lu, X.M. et al., J. Immunol. Methods, 156: 8599, 1992). For protein binding, tetravalent tin and chlorine compound e6 (2 mg / ml) was mixed for 30 minutes at ambient temperature in dimethylformamide with a 10-fold molar excess of EDC (N'-3-dimethylaminopropyl-N-ethylcarbodiimide, hydrochloride, Sigma) . and NHS (N-hydroxysuccinimide, Sigma). The resulting activated mixture was then added to an 8-fold volume of protein solution (1 mg / ml) and incubated at ambient temperature for 1 hour.

After labeling, antibody conjugates were separated from unincorporated fluorophore or photosensitizer using PD-10 columns (Amersham Pharmacia Biotech) equilibrated in 50 mM phosphate, pH 7.4, 100 mM NaCl, (PBS). Immunoreactivity of antibody conjugates was measured as described in the previous example.

In photodestruction experiments, rabbits were administered intravenously with 12 mg of scFv (L19) i-tetravalent tin and chlorine compound e6 (0.8> or 38 mg ovalbumin - tetravalent tin and chlorine compound Θ6 (0.36), and rabbits were kept in 8 hours after injection, rabbits were anesthetized with ketamine (35 mg / kg / xylazine (5 mg / kg) / acepromazine (1 mg / kg)) and one of the two eyes was irradiated for 13 minutes with Schott KL 1500 tungsten halogen lamp equipped with Cy5 excitation filter (Chromá) and two light channels, the ends of which were placed approximately 1 cm from the eye.

The irradiated area was approximately 1 cm in size and the irradiation was 2 with a dose of 100 mW / cm as measured by an SL818 photodetector (Newport Corp. Irvine, CA, USA). No signs of discomfort after irradiation were observed. As a precaution, rabbits received analgesics after irradiation (buprenorphine 0.03 mg / kg). To monitor photodestruction, the eyes were examined with an ophthalmoscope and were photographed using a KOWA SL-14 fundus camera (GMP SA, Rennens, Lausanne, Switzerland). Five rabbits were treated with a conjugate containing a tetravalent tin-chlorine compound ε6 and irradiated to only one eye, while the other eye served as an internal negative control. As an additional control, two rabbits were used that were only irradiated but not conjugated to a photosensitizer.

Immediately after killing rabbits with an overdose of anesthetics, the bulbs were enucleated, the corneas were removed, immersed in Tissue Tek (Sakura Finetechnical, Tokyo, Japan) and frozen. For ex vivo immunofluorescence testing and for some H / E staining, the corneas were fixed in 4% paraformaldehyde in PBS before being immersed in Tissue Tek. Cryostate sections of 5 gm diameter were used for further microscopic analysis. Fluorescent TUNEL tests were performed according to the manufacturer's instructions (Roechem Diagnostic, Rotkreutz, Switzerland).

• φ φφφ φφ ··· ···

Table 1: Sequences of selected anti-ED-B antibody clones

VH string VL string Clone 31-33 * 50-54 * 95-98 * 32 * 50 * 91-96 * A2 SYA AISGSG GLSI Y G NGWYPW G4 SYA AISGSG SFSF Y G GGWLPY El SYA AISGSG FPFY Y G TGRIPP H10 SFS SIRGSS FPFY Y G TGRIPP LI 9 SFS SIRGSS FPFY Y Y TGRIPP Relevant amino acid positions (*: numbering according to Tomlinson et et al., 1995, EMBO J. 14: 4628 -4638) antibody

clones isolated from designed synthetic libraries. Single letter amino acid codes are used according to standard IUPAC nomenclature.

the··

Table 2: Affinities anti-ED-B scFv fragments Clone con (with ' ¹ M' ¹ ) koff (s ¹ ) ^B koff (s ¹ ) ^c K _d (M) * A2 1.5 χ 10 ⁵ 2.8 x 10 ' ³ - 1.9 x 10 ^sec G4 4.0 x 10 " 3.5 x IO ^"3 - 8.7 x 10 ⁸ El 1.6 χ 10 ⁵ 6.5 x IC ³ - 4.1 x 10 ' ⁸ H10 6.7 χ IO ⁴ 5.6 x 10- ⁴ 9.9 x 10 ' ⁵ 1.5 x 10 ' L19 1.1 χ 10 ⁵ 9.6 x 10 ' ⁵ 6.0 x 10 ' ⁶ 5.4 χ 10- *) Kd = koff / con. For H10 high-affinity ligands and L19 are not

_The FF values for BIAcore analysis of the insufficiently precise because of influence negatively-charged carboxylated solid dextran matrix; Kd values are therefore calculated from measurements of K _O ff obtained in competition experiments (see Examples). k _oo , kinetic dissociation constant; k _he , kinetic association constant; kd, dissociation constant. B = measurement on BIAcore; C = measurement by competition with electrochemiluminescence detection. The values are accurate for +/- 50%, based on accurate concentration determination.

Claims (24)

Patent claims An antibody with a specific affinity for a characteristic epitope of the ED-B domain of fibronectin, wherein said antibody has an affinity for said ED-B epitope at subnanomolar concentrations. The antibody of claim 1, wherein said antibody recognizes ED-B (+) fibronectin. The antibody of claim 1, wherein said antibody is in a scFv format. The antibody of claim 3, wherein said antibody is a recombinant antibody. The antibody of claim 3, wherein the affinity is improved by inserting a limited number of mutations in its CDR residues. The antibody of claim 5, wherein the residues are residues 31-33, 50 and 52 and 54 of the VH and the two residues 32 and 50 of the VL domain that have been mutated. The antibody of claim 1, wherein said antibody binds to the ED-B domain of fibronectin with a K _{d of} 54 pM. The antibody of claim 1 having the following amino acid sequence: VH SQASGFTFS SFSMSWVRQA PGKGLEWVSS ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF PYFDYWGQGT LVTVSS (SEQIDNQ: 19) 4 4 connecting group GDGSSQGSGGASTG (SEQ ID NO: 20) VL eivltqspgt lslspgerat LSCRASGSVS ssylawyqqk pgqaprlljy yassratgip DRFSGSGSGT DFTLTISRLE PEPFAVYYCQ QTGRIPPTFG QGTKVEIK (SEQ ID NO: 21) The antibody of claim 1, wherein said antibody is a functionally equivalent variant of L19. The antibody of claim wherein said antibody is radiolabeled. The antibody of claim 10, wherein said antibody is labeled with radioactive iodine. Use of an antibody with specific affinity for a characteristic epitope of the ED-B domain of fibronectin having improved affinity for said ED-B domain for rapid targeted targeting of angiogenesis markers. Use according to claim 12, for the immunoscintigraphic detection of angiogenesis. Use according to claim 13 for detecting diseases characterized by vascular proliferation, such as diabetic retinopathy, age-related macular degeneration or tumors. «· 1 · Use according to claim 12, wherein the antibody locates the respective tissues within three to four hours, preferably within 3 hours, after injection. A diagnostic kit comprising an antibody with specific affinity for a characteristic epitope of the ED-B domain of fibronectin, wherein said antibody has improved affinity for said ED-B domain, and one or more reagents necessary for detecting angiogenesis. Use of an antibody with specific affinity for a characteristic epitope of the ED-B domain of fibronectin, wherein said antibody has improved affinity for said ED-B domain, for the diagnosis and therapy of tumors and diseases characterized by vascular proliferation. 18. Conjugates comprising the antibody of claim 1 and a molecule capable of inducing blood coagulation and blood vessel occlusion. 19. The conjugate of claim 18 wherein the molecule capable of inducing blood coagulation and blood vessel occlusion is a photoactive molecule. 20. The conjugates of claim 19 wherein the photoactive molecule is a photosensitizing agent. The conjugates of claim 20, wherein the photosensitizing agent absorbs light at a wavelength greater than 600 nm. Conjugates according to claim 21, characterized in that the photosensitizing agent is a derivative of a tetravalent tin compound with chlorine eg. Use of the conjugate of claim 18 for the preparation of an injectable preparation for the treatment of pathological conditions associated with angiogenesis. The use of claim 23, wherein the pathological condition associated with angiogenesis is caused by or associated with ocular angiogenesis.